PROJECT SUMMARY Recent studies demonstrate the importance of chronic low-grade inflammation for the development of many devastating diseases such as atherosclerosis, obesity and diabetes. The critical, yet still unclear, step of chronic inflammation is macrophage accumulation in the inflamed peripheral tissue. The overall goal of this proposal is to test the contribution of ?2 integrins to the retention and egress of macrophages within the site of inflammation, specifically during atherogenesis and obesity-induced diabetes. The objective of this work is to evaluate how different surface densities of related integrins ?M?2 and ?D?2, that have similar ligand binding properties, but an opposite effect on the development of inflammation, regulate the accumulation of macrophages in inflamed tissue. The central hypothesis is that the opposing contributions of integrins ?M?2 and ?D?2 to chronic inflammation depend on the different levels of expression of these integrins on the inflammatory macrophages. While a moderate density of ?M?2 supports macrophage egress from inflamed tissue, a high density of ?D?2 promotes macrophage retention within the site of inflammation. Guided by strong preliminary data, this hypothesis will be tested by pursuing three Specific Aims: 1. Evaluate how the levels of ?M and ?D expression at the sites of inflammation regulate the impact of ?M?2 and ?D?2 on the development of metabolic syndrome. 2. Identify the contribution of ?M?2 and ?D?2 to migration of M1 and M2 macrophages in vitro and macrophage trafficking in vivo during obesity-induced diabetes and atherogenesis. 3. Evaluate the effect of short ?D segment on ?D?2-mediated migration in vitro and macrophage retention in adipose tissue and atherosclerotic lesions in vivo. Under the first aim, the role of ?M- and ?D-deficiency on the development of metabolic syndrome will be examined. This data will be supported by the analysis of ?M and ?D expression at the site of inflammation and the mechanism of its regulation on M1- and M2-activated macrophages. Under aim two, the migration of ?D-/- and ?M-/- macrophages will be evaluated using in vitro and in vivo approaches. Under aim three, the potential mechanism of inhibition of ?D-mediated retention of macrophages will be studied using in vitro adhesion and migration assays and in vivo trafficking of adoptively transferred monocytes. The significance of these studies resides in providing new insights on the development of chronic inflammation focusing on the mechanism of macrophage retention, instead of studying macrophage migration to the inflammatory site. This proposal is innovative because the importance of integrin density on the cell surface for the progression and resolution of chronic inflammation has not been previously suggested. Hence, the hypothesis of integrin-dependent retention of macrophages at the site of inflammation proposes a qualitatively new approach for the treatment of chronic inflammatory diseases. Thus, the blocking of integrin ?D during atherogenesis or diabetes can prevent macrophage accumulation at the inflammatory site stimulating the resolution of chronic inflammation and thwarting the progression of the disease.